1. Field of the Invention
The present invention is directed to apparatus and methods for increasing and detecting the fluorescence emitted by a molecule using metal-enhanced fluorescence. In particular, the invention describes the use of surface plasmon excitation for excitation of fluorophores near the metal surface and the efficient collection of the emission by coupling into the plasmon resonance and directing towards the detector. More particularly, the present invention makes use of the unique directionality of the plasmon induced fluorescence signal.
2. Background of the Related Art
Many experiments have been done demonstrating that metal particles can enhance fluorescence. Results in the literature show that fluorescence can be excited via the surface plasmon excitation. See Lakowicz, J. R., Radiative decay engineering: biophysical and biomedical applications. Anal Biochem, 2001. 298(1): p. 1–24; Lakowicz, J. R., et al., Radiative decay engineering: 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer. Anal Biochem, 2002. 301(2): p. 261–77. Results in other publications have shown that emission can couple back into the plasmon at discrete angles.
FIG. 1 shows the well-known total internal reflection in fibers, where the refractive index n2>n1. Light approaching the refractive index boundary from the medium of higher refractive index is reflected away from the normal. For small angles of incidence, there is both a reflected ray and a refracted ray. However at some critical angle of incidence, θc, the refracted ray emerges parallel to the surface. For any angle of incidence greater than θc the light is totally internally reflected back into the medium of higher refractive index.
Fluorescent effects can be excited using various light sources. One light source is Xenon arc lamp. However, the spectral characteristics of the lamp vary over time and cannot provide a stable output. Light-Emitting-Diodes (LED) sources are also used. The LED sources can provide a stable output and offer opportunities for miniaturization and tunability of the output wavelength.